Analysis of Complex DNA Rearrangements during Early Stages of HAC Formation.

Human artificial chromosomes (HACs) are important tools for epigenetic engineering, for measuring chromosome instability (CIN), and for possible gene therapy. However, their use in the latter is potentially limited because the input HAC-seeding DNA can undergo an unpredictable series of rearrangements during HAC formation. As a result, after transfection and HAC formation, each cell clone contains a HAC with a unique structure that cannot be precisely predicted from the structure of the HAC-seeding DNA. Although it has been reported that these rearrangements can happen, the timing and mechanism of their formation has yet to be described. Here we synthesized a HAC-seeding DNA with two distinct structural domains and introduced it into HT1080 cells. We characterized a number of HAC-containing clones and subclones to track DNA rearrangements during HAC establishment. We demonstrated that rearrangements can occur early during HAC formation. Subsequently, the established HAC genomic organization is stably maintained across many cell generations. Thus, early stages in HAC formation appear to at least occasionally involve a process of DNA shredding and shuffling that resembles chromothripsis, an important hallmark of many cancer types. Understanding these events during HAC formation has critical implications for future efforts aimed at synthesizing and exploiting synthetic human chromosomes.

One end to rule them all: Non-homologous end-joining and homologous recombination at DNA double-strand breaks.

Double-strand breaks (DSBs) represent the most severe type of DNA damage since they can lead to genomic rearrangements, events that can initiate and promote tumorigenic processes. DSBs arise from various exogenous agents that induce two single-strand breaks at opposite locations in the DNA double helix. Such two-ended DSBs are repaired in mammalian cells by one of two conceptually different processes, non-homologous end-joining (NHEJ) and homologous recombination (HR). NHEJ has the potential to form rearrangements while HR is believed to be error-free since it uses a homologous template for repair. DSBs can also arise from single-stranded DNA lesions if they lead to replication fork collapse. Such DSBs, however, have only one end and are repaired by HR and not by NHEJ. In fact, the majority of spontaneously arising DSBs are one-ended and HR has likely evolved to repair one-ended DSBs. HR of such DSBs demands the engagement of a second break end that is generated by an approaching replication fork. This HR process can cause rearrangements if a homologous template other than the sister chromatid is used. Thus, both NHEJ and HR have the potential to form rearrangements and the proper choice between them is governed by various factors, including cell cycle phase and genomic location of the lesion. We propose that the specific requirements for repairing one-ended DSBs have shaped HR in a way which makes NHEJ the better choice for the repair of some but not all two-ended DSBs.

Mesenchymal Tumors with EWSR1 Gene Rearrangements.

Among the various genes that can be rearranged in soft tissue neoplasms associated with nonrandom chromosomal translocations, EWSR1 is the most frequent one to partner with other genes to generate recurrent fusion genes. This leads to a spectrum of clinically and pathologically diverse mesenchymal and nonmesenchymal neoplasms, variably manifesting as small round cell, spindle cell, clear cell or adipocytic tumors, or tumors with distinctive myxoid stroma. This review summarizes the growing list of mesenchymal neoplasms that are associated with EWSR1 gene rearrangements.

Chromosome fusions triggered by noncoding RNA.

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in 4 distinct cases (out of 4 tested), including one fusion of 3 chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of 2 distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

Primary pulmonary clear cell sarcoma-the first two reported cases.

We report two cases (male patients 50 and 55 years old) of clear cell sarcoma ("melanoma of soft parts") arising in the lung, of which one case showed regional lymph node metastases. Histologically, both tumors displayed varying clear epithelioid and spindle neoplastic cells arranged in storiform and nested growth patterns, separated by thin fibrovascular septa. Immunohistochemical studies demonstrated positive expression of S-100 protein, HMB-45 and Melan-A in one case and S-100 protein only in the other. Fluorescence in situ hybridization showed positive EWSR1 gene rearrangement, and a presence of EWS-ATF1 fusion transcript was confirmed by RT-PCR and sequencing in one case.

NSCLC: An Update of Driver Mutations, Their Role in Pathogenesis and Clinical Significance.

Lung cancer is the most common malignancy in the US and causes the most cancer-related deaths. Non-small-cell lung carcinoma (NSCLC) accounts for the majority of cases. NSCLC historically was considered one entity, reflected by platinum-based therapy as the standard of care; however, with the discovery of EGFR mutations and ALK rearrangements, the landscape of treatment has become more personalized reflecting genomic heterogeneity. The molecular basis for tumor genesis was recognized and became a new method of classification. The availability of tumor sequencing and testing for these mutations is also becoming more accessible outside of major academic institutions. Targeted therapies offer alternatives to dangerous cytotoxic chemotherapy with equal or better efficacy. With these changes, driver mutations will play an increasing role in the diagnosis and treatment of NSCLC. In this review we will examine the characteristics of several NSCLC driver mutations and gene rearrangements and emerging data on therapies directed against them.

ALK and ROS1 rearrangements tested by fluorescence in situ hybridization in cytological smears from advanced non-small cell lung cancer patients.

BACKGROUND: The identification of ALK and ROS1 rearrangements and the availability of an effective target therapy, such as crizotinib, represent a new option in the treatment of advanced non-small cell lung cancer (NSCLC) patients. In light of recent advances in non-invasive diagnostic procedures, we aimed to demonstrate that direct cytological smears are suitable for assessing ALK and ROS1 rearrangements in patients with NSCLC. METHODS: Fifty-five patients with a cytological diagnosis of lung adenocarcinoma (ADC) were evaluated for ALK rearrangements by fluorescence in situ hybridization (FISH) and 12 patients for ROS1 FISH rearrangements. Seventeen of the 55 cytological samples tested for ALK were obtained from the primary tumor and 38 from metastatic lesions. Ten of 12 samples evaluated for ROS1 were obtained from metastatic sites and two from the primary tumor. RESULTS: ALK FISH was successful in 49/55 (89%) cytological ADC samples and ROS1 FISH in all 12 cytological samples. ALK rearrangements were found in 3/13 (23%) primary tumors and 7/36 (19%) metastatic sites. ROS1 rearrangements were found in one of the two primary tumors and in two of the 10 metastases. Two of the three rearranged cases were tested on cytology after knowing that they were rearranged on histology in order to increase representativeness of ROS1 rearranged cases in this study. CONCLUSION: Whenever cytology represents the only available material for diagnosis and biological characterization of NSCLC, minimally invasive procedures may provide an additional important source of cellular material for FISH assessment of ALK and ROS1 rearrangements.

The "next-generation" knowledge of papillary thyroid carcinoma.

The application of Next-Generation Sequencing for studying the genetics of papillary thyroid carcinomas (PTC) has recently revealed new somatic mutations and gene fusions as potential new tumor-initiating events in patients without any known driver lesion. Gene and miRNA expression analyses defined clinically relevant subclasses correlated to tumor progression. In addition, it has been shown that tumor driver mutations in BRAF, and RET rearrangements - altogether termed "BRAF-like" carcinomas - have a very similar expression pattern and constitute a distinct category. Conversely, "RAS-like" carcinomas have a different genomic, epigenomic, and proteomic profile. These findings justify the need to reconsider PTC classification schemes.

Inflammatory myofibroblastic tumour of the urinary bladder: the role of immunoglobulin G4 and the comparison of two immunohistochemical antibodies and fluorescence in-situ hybridization for the detection of anaplastic lymphoma kinase alterations.

AIMS: We examined gene rearrangement and the expression of anaplastic lymphoma kinase (ALK) in urinary bladder inflammatory myofibroblastic tumour (IMT) using fluorescence in-situ hybridization (FISH) and two immunohistochemical antibodies to ALK. We also investigated whether IMT represents an immunoglobulin (Ig)G4-related disease. METHODS AND RESULTS: The performance of the Dako FLEX ALK monoclonal antibody (CD246) and the Cell Signaling Technology ALK (D5F3) XP monoclonal antibody were compared. Overall, 11 of 16 tumours showed ALK expression by immunohistochemistry (69%). Ten demonstrated ALK expression with both stains and one was positive with D5F3 but not CD246 (91% correlation). The D5F3 antibody yielded a stronger staining intensity and a higher sensitivity. Nine tumours demonstrated ALK rearrangements (56%) by FISH. Three were ALK(+) by immunohistochemistry but negative for rearrangement by FISH, whereas one showed rearrangement by FISH but was negative by immunohistochemistry. In total, 12 tumours were positive for ALK abnormalities (75%). Using current criteria, no cases were classified as an IgG4-related disease. CONCLUSIONS: The ALK D5F3 immunohistochemical stain showed superior staining characteristics compared with ALK CD246. Discrepancies in the results between FISH and immunohistochemistry for ALK abnormalities may have causes that are multifactorial. By current criteria, IMT does not represent an IgG4-related disease.

NUT midline carcinoma of the mediastinum showing two types of poorly differentiated tumor cells: a case report and a literature review.

Nuclear protein in testis (NUT) midline carcinoma (NMC) is an extremely aggressive carcinoma that is genetically defined by rearrangement of the NUT gene. Herein, we describe a case of NMC in a young Japanese man, and review 31 cases of NMC in the literature. The present case was of a massive tumor of the anterior and middle mediastinum in a 26-year-old man. The tumor included 2 types of poorly differentiated tumor cells and was immunohistochemically positive for the NUT-specific antigen, epithelial membrane antigen (EMA), cluster of differentiation 99 (CD99) antigen, CD45RO antigen, keratins, p63, and p40. The patient died 2 months after the initial diagnosis. At least two-thirds of the 31 NMC cases in the literature were immunohistochemically positive for EMA, p63, and AE1/AE3. However, some exceptional NMC cases are keratins-negative/CD99-positive like Ewing sarcoma or CD45RO-positive like the present case.

Chromosome 8q24.1/c-MYC abnormality: a marker for high-risk myeloma.

The proto-oncogene c-MYC is rearranged in about 15% of patients with multiple myeloma (MM). We identified 23 patients with MM and c-MYC. Primary objectives were to describe the clinical characteristics, response to therapy, progression-free survival and overall survival (OS). Twelve out of twenty-three patients presented with or progressed to either plasma cell leukemia (PCL) and/or extramedullary disease (EMD). Induction therapy consisted of an immunomodulatory, proteasome inhibitor-based or conventional chemotherapy regimen. Fifteen patients achieved a partial response and three achieved a very good partial response. Sixteen patients received an autologous and one patient an allogeneic hematopoietic stem cell transplant. Median OS from diagnosis was 20.2 months. Patients with PCL or EMD had significantly shorter OS (15.5 vs. 40.4 months, p = 0.0005). This is the first report describing the clinical characteristics of patients with MM and c-MYC. These abnormalities are associated with an aggressive form of MM, high incidence of PCL/EMD and short OS.

Erythropoiesis and globin switching in compound Klf1::Bcl11a mutant mice.

B-cell lymphoma 11A (BCL11A) downregulation in human primary adult erythroid progenitors results in elevated expression of fetal γ-globin. Recent reports showed that BCL11A expression is activated by KLF1, leading to γ-globin repression. To study regulation of erythropoiesis and globin expression by KLF1 and BCL11A in an in vivo model, we used mice carrying a human ß-globin locus transgene with combinations of Klf1 knockout, Bcl11a floxed, and EpoR(Cre) knockin alleles. We found a higher percentage of reticulocytes in adult Klf1(wt/ko) mice and a mild compensated anemia in Bcl11a(cko/cko) mice. These phenotypes were more pronounced in compound Klf1(wt/ko)::Bcl11a(cko/cko) mice. Analysis of Klf1(wt/ko), Bcl11a(cko/cko), and Klf1(wt/ko)::Bcl11a(cko/cko) mutant embryos demonstrated increased expression of mouse embryonic globins during fetal development. Expression of human γ-globin remained high in Bcl11a(cko/cko) embryos during fetal development, and this was further augmented in Klf1(wt/ko)::Bcl11a(cko/cko) embryos. After birth, expression of human γ-globin and mouse embryonic globins decreased in Bcl11a(cko/cko) and Klf1(wt/ko)::Bcl11a(cko/cko) mice, but the levels remained much higher than those observed in control animals. Collectively, our data support an important role for the KLF1-BCL11A axis in erythroid maturation and developmental regulation of globin expression.

Expression of mutated IGHV3-23 genes in chronic lymphocytic leukemia identifies a disease subset with peculiar clinical and biological features.

PURPOSE: B-cell chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease whose outcome can be foreseen by investigating the mutational status of immunoglobulin heavy chain variable (IGHV) genes. Moreover, a different prognosis was reported for CLL expressing specific IGHV genes in the context or not of stereotyped B-cell receptors. Here we investigated novel associations between usage of specific IGHV genes and clinical features in CLL. EXPERIMENTAL DESIGN: Among 1,426 CLL-specific IG-rearrangements, stereotyped B-cell receptor clusters never utilized the IGHV3-23 gene. Given this notion, this study was aimed at characterizing the IGHV3-23 gene in CLL, and identifying the properties of IGHV3-23-expressing CLL. RESULTS: IGHV3-23 was the second most frequently used (134 of 1,426) and usually mutated (M; 109 of 134) IGHV gene in our CLL series. In the vast majority of M IGHV3-23 sequences, the configuration of the 13 amino acids involved in superantigen recognition was consistent with superantigen binding. Clinically, M IGHV3-23 CLL had shorter time-to-treatment than other M non-IGHV3-23 CLL, and multivariate analyses selected IGHV3-23 gene usage, Rai staging, and chromosomal abnormalities as independent prognosticators for M CLL. Compared with M non-IGHV3-23 CLL, the gene expression profile of M IGHV3-23 CLL was deprived in genes, including the growth/tumor suppressor genes PDCD4, TIA1, and RASSF5, whose downregulation is under control of miR-15a and miR-16-1. Accordingly, relatively higher levels of miR-15a and miR-16-1 were found in M IGHV3-23 compared with M non-IGHV3-23 CLL. CONCLUSIONS: Altogether, expression of the IGHV3-23 gene characterizes a CLL subset with distinct clinical and biological features.

T-cell lymphoblastic leukemia in early childhood presents NOTCH1 mutations and MLL rearrangements.

T-cell acute lymphoblastic leukemia (T-ALL) may affect children in very early age. However, the critical events leading to this brief latency is still unclear. We used standard methods to explore NOTCH1 mutations and other specific molecular markers in 15 early childhood T-ALL cases. Most of them consisted of immature differentiation subtype. Despite being found in a lower frequency than that described for overall pediatric T-ALL, NOTCH1 alterations were the most frequent ones. Other alterations included MLL(+) (n=4), SIL-TAL1(+) (n=3), FLT3 mutation (n=1) and HOX11L2(+) (n=1). Our results suggest that NOTCH1 and MLL abnormalities are primary leukemogenic hits in early T-ALL.

The replication fork's five degrees of freedom, their failure and genome rearrangements.

Genome rearrangements are important in pathology and evolution. The thesis of this review is that the genome is in peril when replication forks stall, and stalled forks are normally rescued by error-free mechanisms. Failure of error-free mechanisms results in large-scale chromosome changes called gross chromosomal rearrangements, GCRs, by the aficionados. In this review we discuss five error-free mechanisms a replication fork may use to overcome blockage, mechanisms that are still poorly understood. We then speculate on how genome rearrangements may occur when such mechanisms fail. Replication fork recovery failure may be an important feature of the oncogenic process. (Feedback to the authors on topics discussed herein is welcome.).

NKT cells: from totipotency to regenerative medicine.

The recent discovery that natural killer T (NKT) cell nuclei are totipotent opens a novel avenue for further understanding NKT cell function in normal and diseased states. The progeny of a cloned mouse harboring the in-frame rearranged Valpha14-Jalpha18 T cell receptor in one allele showed a significant increase in NKT cell number compared with wild-type or littermate control mice that possessed a different TCR. Importantly, NKT cells from such progeny produced both interferon-gamma and interleukin-4, a hallmark of NKT cells. In these progeny, NKT cell development appeared to be instructively, rather than permissively, determined. Using embryonic stem cells prepared via the somatic cell nuclear transfer of NKT nuclei, relatively mature NKT cells were induced under conditions permissible for T cell induction. Furthermore, these NKT cells matured autonomously upon injection into mice, resulting in an antigen-specific adjuvant effect.

NMD is essential for hematopoietic stem and progenitor cells and for eliminating by-products of programmed DNA rearrangements.

Nonsense-mediated mRNA decay (NMD) is a post-transcriptional surveillance process that eliminates mRNAs containing premature termination codons (PTCs). NMD has been hypothesized to impact on several aspects of cellular function; however, its importance in the context of a mammalian organism has not been addressed in detail. Here we use mouse genetics to demonstrate that hematopoietic-specific deletion of Upf2, a core NMD factor, led to the rapid, complete, and lasting cell-autonomous extinction of all hematopoietic stem and progenitor populations. In contrast, more differentiated cells were only mildly affected in Upf2-null mice, suggesting that NMD is mainly essential for proliferating cells. Furthermore, we show that UPF2 loss resulted in the accumulation of nonproductive rearrangement by-products from the Tcrb locus and that this, as opposed to the general loss of NMD, was particularly detrimental to developing T-cells. At the molecular level, gene expression analysis showed that Upf2 deletion led to a profound skewing toward up-regulated mRNAs, highly enriched in transcripts derived from processed pseudogenes, and that NMD impacts on regulated alternative splicing events. Collectively, our data demonstrate a unique requirement of NMD for organismal survival.

The endless tale of non-homologous end-joining.

DNA double-strand breaks (DSBs) are introduced in cells by ionizing radiation and reactive oxygen species. In addition, they are commonly generated during V(D)J recombination, an essential aspect of the developing immune system. Failure to effectively repair these DSBs can result in chromosome breakage, cell death, onset of cancer, and defects in the immune system of higher vertebrates. Fortunately, all mammalian cells possess two enzymatic pathways that mediate the repair of DSBs: homologous recombination and non-homologous end-joining (NHEJ). The NHEJ process utilizes enzymes that capture both ends of the broken DNA molecule, bring them together in a synaptic DNA-protein complex, and finally repair the DNA break. In this review, all the known enzymes that play a role in the NHEJ process are discussed and a working model for the co-operation of these enzymes during DSB repair is presented.